Structural features of the vesicle of sp. CpIl in culture

JOHNG. TORREYAND DALECALLAHAM Cabot Foundation, Harvard University, Petersham, MA, U.S.A.01366 and Department of Botany, University of Massachusetts, Amherst, MA, U.S.A.01003 Accepted March 16, 1982

TORREY,J. G., and D. CALLAHAM.1982. Structural features of the vesicle of Frankia sp. CpII inculture. Can. J. Microbiol. 28: 749-757. The filamentous bacterium Frankia sp. CpIl of the , responsible for symbiotic in the nodules of certain woody dicots, also fixes dinitrogen when grown independently of the host in a nitrogen-free synthetic nutrient medium under aerobic conditions. In structural studies of Frankla grown in culture it has been shown that the bacterial filaments form vesicles, enlarged terminal endings in which the enzyme is formed. Microscopic examination of cultures shows that the vesicles possess a specialized envelope consisting of a number of thin layers or laminae which In polarized light show birefringence and in freeze-etch electron microscopy are resolved as multiple (12-15) laminae approximately 35-40 A (1 A = 0.1 nm) in thickness. Comparisons are made between the structure of the veslcle envelope in cultured Frankia and the ; strikingly similar innermost laminated layer in the dinitrogen-fixing heterocysts of the cyanobacterium Anabaena. Comparable protective functions in limiting oxygen to the dinitrogen-fixing sites are suggested for these similar structures in two quite unrelated microorganisms. 1 TORREY,J. G., et D. CALLAHAM.1982. Structural features of the vesicle of Frankia sp. CpIl in culture. Can. J. Microbiol. 28: 749-757. I La bactkrie filamenteuse Frankia sp. CpII du groupe ActinomycCtales, responsable de la fixation symbiotique de l'azote dans les nodules de certaines dicotylCdones ligneuses, fixe Cgalement l'azote libre lorsqu'elle croit indkpendament de l'hdte sur un milieu nutritif synthCtique dCpouwu d'azote en condition aCrobique. Dans des Ctudes structurales de Frankia en culture, on a pu verifier que les filaments bactCriens foment des vCsicules par gonflement des extrCmitCs dans lesquelles l'enzyme nitrogknase est fomCe. L'examen microscopique des cultures fait ressortir que les vCsicules posZdent une enveloppe spCciallsCe constituCe d'un certain nombre de couches fines ou feuillets qui, en lumikre polariste, prCsentent de la birCfnngence et que la microscopie Clectronique, par dCcapage a froid, rCsout comedes feuillets multiples (12- 15) d'environ 35-40 A (1 A = 0,l nm) dlCpaisseur. Des comparaisons sont Ctablies entre la structure de I'enveloppe vtsiculaire de Frankia en culture et la couche laminee la plus For personal use only. interne, trks semblable, des hCtCrocystes fixateurs d'azote de la cyanobactkrie Anabaena. I1 est alors suggCrC que ces structures qui se ressemblent, bien que appartenant a deux organismes non reliCs, exercent des fonctions de protection comparables en limitant I'oxygkne dans les sites fixateurs d'azote libre. [Traduit par le journal] Introduction 1964; Lalonde and Knowles 1975a; Newcomb et al. One of the striking structural features of the 1978). In freeze-etch preparations of root nodules of involving the actinomycete Frankia within the root Alnus, Lalonde and Devoe (1976) and Lalonde et al. nodules of all actinorhizal plants thus far studied is the (1976) showed that this space disappeared and that one presence of a polysaccharide encapsulation synthesized could account for all the layers continuously between by the host cells and laid down around every filament, actinomycete and host cytoplasm as membranes of the sporangium, and vesicle of the invasive organism bacterium, host cell, or encapsulation. (Lalonde and Knowles 1975a, 1975b; Newcomb et al. The enzyme nitrogenase when exposed to molecular 1978). The polysaccharide is presumed to be pectic in oxygen is labile; this general characteristic of nitrogen- nature (Lalonde and Knowles 1975b) and is synthesized ase observed in all in vitro preparations is equally true of and assembled by host cells in the accommodation of the the nitrogenase from Frankia (Benson et al. 1979). microbial associate from the outset of the infection Within the bacteroids of leguminous root nodules the (Callaham et al. 1979). nitrogenase is maintained at a low Po2 by structural In all transmission electron micrographs published of modifications of the nodule and by the presence of Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 nodule structure of the actinomycetes one observes leghaemoglobin which is produced by the host cells (cf. : clear zones between the actinomycete and the host Tjepkema 1979). Haemoglobinlike compounds have cytoplasm, an area believed to be an artefact of fixation been reported in actinorhizal root nodules (Davenport (Lalonde et al. 1976). This clear zone is particularly 1960) but these observations have not been confirmed by prominent around vesicles in nodules (Becking et al. others (cf. Bond 1974). Tjepkema (1979) showed that 0008-4 166/82/070749-09$01 .OO/O 01982 National Research Council of Canada/Conseil national de recherches du Canada 750 CAN. J. MICROBIOL. VOL. 28. 1982

molecular oxygen is freely diffusible to the nodule cells Glutaraldehyde - osmium tetroxide furation containing the actinomycetal endophyte filaments. The Filaments of CpIl bearing vesicles were harvested and polysaccharide capsule cannot be expected to provide a suspended in culture medium containing 2% glutaraldehyde barrier to oxygen diffusion. The nature of the protection and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 6.8, of nitrogenase within the actinorhizal nodule from for 12 h at 4°C. Cultures were washed in 0.1 M cacodylate buffer and postfixed in 2% osmium tetroxide in the same oxygen destruction remains to be determined. buffer. Cultures were washed three times with water, dehy- Induction of vesicle formation occurs when a filamen- drated in a graded alcohol series, and embedded in Epon- tous culture of Frankia sp. CpIl is subcultured into a Araldite resin. defined medium lacking fixed nitrogen substrates and Glutaraldehyde - potassium permanganate (KMn04)fura- containing succinate and EDTA (Tjepkema et al. 1980). tion Concomitant with vesicle formation in vitro one can Filaments of CpIl were harvested and fixed in glutaralde- demonstrate the onset of acetylene-reducing activity hyde-paraformaldehyde as described above and then fixed which increases with age of culture paralleling the with 2% aqueous potassium permanganate for 12 h at 4°C increase in the number of vesicles formed. The activity followed by washing, dehydration, and embedding as of the enzyme nitrogenase formed within the vesicles is described above. Glutaraldehyde - periodic acid - thiocarbohydrazide relatively unaffected by ambient values in culture O2 furation and is sustained even up to approximately 40% O2in the Cultures were fixed in glutaraldehyde-paraformaldehyde as atmosphere (Tjepkema et al. 1980, 1981). The nitro- described above and washed three times with distilled water. genase activity of Frankia vesicles produced in vitro The cells were then treated for 30 min with 2% periodic acid at suggests that the vesicles themselves provide the mech- room temperature, washed, treated with 1% osmium tetroxide anism to protect the enzyme within the vesicle from at pH 6.8 in 0.05 M cacodylate buffer for 1 h at 23"C, washed denaturation by molecular oxygen. For these reasons the in water, treated with 0.2% thiocarbohydrazide in 20% acetic structure of the vesicle envelope has become of great acid for 30 min, washed with 20, 10, and 5% acetic acid and interest and was the focus of the study reported here. then water, treated 30 min with 1% osmium tetroxide in 0.05 M cacodylate buffer, pH 6.8, washed four times in Materials and methods distilled water, and dehydrated and embedded as described above. Light microscopy Cultures of Frankia sp. CpIl were maintained in liquid Freeze-etch preparations nutrient culture and induced to form nitrogen-fixing vesicles as Cultures of CpIl were harvested and resuspended in culture described by Tjepkema et al. (1981). Living or glutaralde- medium made up to 20% with glycerol for 1 h. Each culture was then pelleted and small bits of CpIl were mounted on gold

For personal use only. hyde-fixed cultures were photographed using phase-contrast, Nomarski differential interference contrast, or polarizing specimen supports and plunged into Freon-22 at its freezing optics with a Reichert Zetopan photomicroscope. Measure- point. Frozen specimens were fractured, etched, shadowed ment of polarized light retardation by the vesicle was made with platinum-carbon, and replicated with carbon in a Balzers using a A130 Kohler compensator (Zeiss). freeze-etch apparatus. Specimens were etched for 30-60 s with the stage at - 100°C. Thin sections of CpIl vesicles from Electron microscopy culture and freeze-etch replicas of vesicles were photographed Tissues of root nodules of Comptoniaperegrina (L.) Coult. on a JEOL 100 CX electron microscope. were prepared for transmission electron microscopy as de- scribed by Newcomb et al. (1978). Results Fixation Observations on nodule cells: light microscopy Vesicles of CpIl produced in vitro were prepared for Glutaraldehyde fixation followed by postfixation transmission electron microscopy by several fixation tech- staining, plastic embedment, and sectioning for light niques in attempts to preserve the structure of the vesicle and transmission electron microscopy allow one to envelope. examine the structure of the endosymbiont of actino-

FIG. 1. Septate vesicles of the endophyte of peregrina in a nodule in vivo after fixation with glutaraldehyde postfixed with osmium and stained with uranyl acetate followed by lead citrate. The club-shaped vesicles develop at the perimeter of the infected cells of the maturing region of the nodule cortex. The void areas (arrows) are clear spaces surrounding each vesicle. Vacuoles (va) lined with phenolics occur adjacent to the nodule cell wall (w). Bar = 1 pm FIG. 2. Nomarski Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 micrograph of a filamentous culture of CpIl showing two spherical vesicles (v)developed in vitro. A disrupted sporangium (sp) is located between the vesicles. Note the distinctive wall and envelope of the vesicle as evidenced by Nomarski optics. Bar = 10 pm. FIG. 3. Phase-contrast micrograph of a culture of CpI1. The spherical vesicle on a short stalk appears dark in contrast with the much more strongly refractile spores within or releasing from sporangia which are phase bright. Bar = 10 pm. FIG. 4. Anoptral phase-contrast micrograph of cultured CpIl illustrating a vesicle and a very young sporangium. The vesicle wall shows a phase shift distinct from that produced by the thin-walled immature sporangium. The halo surrounding the vesicle is attributable to the envelope. Bar = 10 pm. TORREY AND CALLAHAM 75 1 For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 752 CAN. J. MICROBIOL. VOL. 28, 1982

rhizal root nodules with some precision. An electron phase shift imparted by the vesicle envelope. Such a micrograph (Fig. 1) of an ultrathin plastic section halo is not seen surrounding the filaments or young stained with uranyl acetate and lead citrate of Frankia sporangium. within cells of Comptonia peregrina shows In an effort to better characterize the nature of the the swollen terminal ends of filaments designated vesicle envelope we examined with polarizing optics vesicles. In Comptonia these vesicles are elongate or living cultures of Frankia sp. CpIl containing vesicles. club shaped (Newcomb et al. 1978); in Alnus the In Figs. 5,6, and 7 is shown the same microscopic field vesicles are more spherical. Typically, they are septate containing a vesicle in culture. The typical Nomarski and show internal differentiation. The shape of the image of Fig. 5 is changed by polarizing filters to a vesicles within nodule cells of different hosts is under striking birefringence (Fig. 6) attributable to the nature the control of the host cells (Lalonde 1978). The vesicles of the vesicle envelope. In Fig. 7 the rotation of the as well as the rest of the Frankia filaments within the compensator filter reduced the birefringence. In Fig. 6 nodule cells are surrounded by and "shaped" by the one can see the "maltese-cross" configuration of the polysaccharide encapsulation laid down by the host outer structure of this spherical body, which can be cells. The clear space adjacent to the endophyte in Fig. 1 interpreted as either a circumferential layering of thin can be attributed in part to the encapsulation. laminae forming the outer layer or envelope of the vesicle or as a radial arrangement of elements compris- Observations on cultured Frankia: light microscopy ing the envelope. The optical basis for such birefrin- When the actinomycete isolated from Comptonia gence has been described (Bennett 1950) and leads us to peregrina, Frankia sp. CpI1, is grown in a defined the conclusion that the vesicle envelope is distinct from medium lacking reduced nitrogen and supplemented other outer membranes of cultured Frankia. In a similar with succinate and EDTA, vesicles are formed in comparison Figs. 8 and 9 show a field of cultured cells of abundance after a few days in culture and their numbers Frankia containing sporangia and vesicles, observed increase with time. Cultures of Frankia containing with (Fig. 9) and without (Fig. 8) crossed polarizer and vesicles effectively reduce acetylene to ethylene in analyzer. The birefringence seen in the vesicle envelope culture approximately in proportion to the number of is clearly lacking in the sporangia and spores. vesicles formed (Tjepkema et al. 1980, 1981). Such cultures have been shown by 15~studies to reduce Electron microscopic observations of cultured cells of dinitrogen (Torrey et al. 1981). Vesicles formed in Frankia culture are globose, vary in size from 3 to 5 pm, and With these insights into the specialized nature of the For personal use only. each is attached as a side branch of a filament with a vesicle envelope we turned toward attempts to visualize vesicle stalk whose length is approximately equal to the the structure of the vesicle in the electron microscope. vesicle diameter. In cultured Frankia, no polysaccha- Efforts to visualize the outer membranes of the vesicle ride encapsulation is present and the outer limits of the using the transmission electron microscope have been filaments, sporangia, and vesicle are determined by the notably unrewarding. Fixation using standard methods membranes and wall layers of the endophyte itself. Our with glutaraldehyde with or without osmium postfixa- efforts were directed to elucidating these outer struc- tion staining has shown that there is a loss in preparation tures, especially in the vesicle. of material from the envelope. In Fig. 10 is illustrated a Examination of living cultures of Frankia sp. CpIl section of a vesicle in which the membranes of the under Nomarski optics (Fig. 2) shows all structures of internal septa have been preserved but which shows only the culture with about equal contrast. Here, a vesicle wisps of enveloping membrane and a halo or space envelope becomes visible, appearing as a thickened wall surrounding the vesicle. If one foregoes preservation of around the swollen vesicle itself. No such envelope is the inner vesicle contents in efforts to preserve the observed along the filaments or around the sporangia. envelope using different fixation and poststaining pro- Living preparations of cultured Frankia sp. CpIl ob- cedures, one loses most structural detail without notable served under normal phase optics (Fig. 3) show the success in preserving the outer layers. In Fig. 11 the filaments and vesicles as phase dark with sporangia and specimen was prepared for transmission electron mi- spores more strongly refractile and phase bright. The croscopy using glutaraldehyde fixation followed by per-

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 nearly spherical vesicle and its stalk attached to a linear manganate. Only remnants of an envelope are seen in filament are clearly evident. With anoptral phase- section and the clear impression is given of a loss of contrast optics (Fig. 4) there is a sharp distinction not material around the vesicle body. With even more evident by ordinary phase-contrast optics. The vesicle is caustic methods, which caused severe disruption of much brighter than the short vesicle stalk, vegetative internal vesicle structure (Fig. 12), some structural filaments, or the young sporangium. The multilayered elements showing lamination were preserved in the halo is an optical effect presumably arising by a large outer enveloping layers, but detail was lost. TORREY AND CALLAHAM For personal use only.

FIGS.5-7. Series of photographs of a vesicle of CpIl formed in vitro. Fig. 5. Nomarski micrograph showing some aspects of structure of the vesicle. Note the distinctly evident envelope of the vesicle. Bar = 10 p-m. Fig. 6. Birefringence of the vesicle envelope viewed through crossed polarizer and analyzer. The cross pattern of birefringence in the vesicle is due either to radial or to circumferential laminar structure in the vesicle wall envelope. Bar = 10 p-m Fig. 7. Polarizing microscope photograph showing the birefringence of the vesicle of Fig. 6 compensated in the +90° axis. Retardation by this vesicle envelope is 14.3nrn. Bar = 10 p-m FIG.8. Vesicle (v) and mature sporangium (sp) of CpIl in culture. Note the distinctive appearance of the vesicle wall and envelope when viewed with Nomarski optics. The mature, thick-walled spores do not give this appearance (see text). Bar = 10 p-m. FIG. 9. Polarized light micrograph of the same field shown in Fig. 8. The vesicle envelope is moderately birefringent while the mature, thick-walled spores show only an extremely weak birefringence. Bar = 10 Km. Freeze-fracture electron microscopy of fresh, living envelope is shown in Fig. 14a. As many as 12- 15 thin material was then attempted. From these preparations laminae can be observed either on fractured face views we were able to observe the laminated structure of the (Fig. 14b) or in occasional places in these photographs vesicle envelope which had been predicted from obser- in section. Calculations of the thickness of the laminae vations based on the optical birefringence in polarized based on these photomicrographs lead to an estimate of light. In Fig. 13 is seen a freeze-fracture preparation of an average thickness of 35-40 A (1 A = 0.1 nm). An an entire vesicle in which the fracture face at the surface end-on view of the base of a vesicle at the position of its Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 of the vesicle peeled away some of the multiple layers of attachment to the stalk is illustrated in Fig. 15. Our the outer vesicle envelope. Since no polysaccharide interpretation is that the stalk also shows laminae which capsule exists in cultured Frankia cells (since the are fewer in number than those surrounding the vesicle encapsulation is a product of the host cell and is only itself and many attenuate toward the base of the vesicle formed in the symbiotic state within the nodule), the stem. We see no evidence of such laminae surrounding multilayered structure observed here is the vesicle spores, sporangia, or vegetative filaments of the en- envelope. Another view of a vesicle with its layered dophyte in freeze-etch preparations. Some attention has ......

CAN. J. MICROBIOL. VOL. 28, 1982

FIG. 10. Electron micrograph of a septate vesicle of CpIl formed on filaments cultured in vitro. The vesicle was fixed by glutaraldehyde and postfixed with osmium. Note the wisp of electron-dense material that encircles the vesicle (arrows). Bar =

For personal use only. 1 ym. FIG. 11. Electron micrograph of CpIl vesicle formed in culture fixed with glutaraldehyde followed by permanganate. Some of the electron-dense enveloping material (arrow) is preserved but appears dispersed and widely separated from the vesicle wall. Bar = 1 ym. been paid in the literature (Lechevalier and Lechevalier organ of the microorganism within which the enzyme 1979; van Dijk and Merkus 1976) to the occurrence nitrogenase functions independently of the host cell and around spores of Frankia of so-called double-track or its specialized encapsulation. It is clear from the in vitro ...... , ...... triple-track membranes, but there is no evidence as to activity of vesicles that host-synthesized polysaccharide ...... - ...- ...... , ...... their function. is not essential to the activity of the enzyme within the vesicle. From the studies presented above, we have concluded that the vesicle formed in the free-living state Discussion possesses an outer envelope composed of a complex Cultivation of Frankia in the free-living state and multilaminate enclosure which surrounds the entire induction of vesicle formation by manipulation of the vesicle and probably extends along the length of the nusents in the culture medium allow one to study the vesicle stalk to its point of attachment to the vegetative structural nature of the vesicle, i.e., the differentiated filament.

FIG. 12. Transmission electron microscope section of a vesicle prepared after glutaraldehyde fixation by sequential treatment Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 with 1% periodic acid, 1% osmium tetroxide, 0.2% thiocarbohydrazide, and 1% osmium tetroxide. The vesicle shows membranous septa (s). Enveloping vesicle layers, best visible at arrows, appear stabilized and remain closely appressed to the vesicle wall. Bar = 0.5 ym FIGS. 13-15. Freeze-etch preparations of vesicles of CpIl produced on filaments cultured in vitro. Fig. 13. Vesicle which has fractured through the vesicle envelope, exposing many layers in face and edge aspects. The hyphal stalk is attached to the vesicle at the bottom of the photograph. Bar = 1 ym. Fig. 14a. Vesicle which has been fractured during freeze-etch preparation to expose the numerous laminae of the vesicle envelope, most clearly evident at the arrows. The hyphal attachment is fractured transversely at the bottom of the vesicle. Bar = 1 ym. Fig. 14b. Enlarged view of laminae of vesicle envelope. Bar = 0.1 ym. Fig. 15. Freeze-etch preparation in which the vesicle has been fractured through an outer edge probably at the base of the vesicle and shows the layered envelope (arrow), the vesicle wall (w), and vesicle cytoplasm (cy). Bar = 0.5 ym. . . TORREY AND CALLAHAM 755 For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 CAN. J. MICROBI(IL. VOL. 28, 1982

From arguments developed below we believe the We have yet to achieve excellent preservation for laminae of the vesicle envelope are probably specialized transmission electron microscopy. lipid layers which are easily washed out by preparative There is strong evidence that the laminated layer in methods used for transmission electron microscopy, heterocysts of are made of glycolipids and leaving a "void area" frequently observed and discussed that these glycolipid layers serve to limit gaseous in the literature. These lipid layers are preserved in exchange into and out of the heterocyst, site of the freeze-fracture procedures and can be visualized. enzyme nitrogenase. Nichols and Wood (1968) first In their studies of Frankia filaments and vesicles in demonstrated the presence in polar lipid fractions root nodules of Alnus, Lalonde and his colleagues extracted from nitrogen-fixing Anabaena of a unique devoted considerable effort to understanding the layers glycolipid component. Subsequently, Winkenbach et surrounding the endophyte within the nodule. The al. (1972) demonstrated that a group of glycolipids multilayered nature of the membranes and encapsulation specific to heterocyst-forming cyanobacteria were local- is particularly difficult to decipher within the nodule; the ized in the laminated layer of the heterocyst envelope. cultured endophyte which has formed vesicles in vitro is Isolation of the laminated layer by fractionation using much simpler. In the freeze-etch micrographs of Alnus sucrose gradients allowed an examination of the lami- nodules, Lalonde and Devoe (1976) and Lalonde et al. nated layer. The multiple lamellae were visualized by (1976) failed to distinguish an encapsulation layer freeze-etch electron microscopy and the isolated frag- outside the laminated layers surrounding the vesicles ments of the lamella were shown to be birefringent in and erroneously designated the laminae themselves as polarized light. Bryce et al. (1972) identified the part of the encapsulation. Since in our photornicro- glycolipids from heterocysts of Anabaena as hexose graphs of vesicles in cultured Frankia the laminae are derivatives of long-chain polyhydroxy alcohols. Their present and in approximately the same numbers as chemical nature was further described by Lambein and observed in the nodule by Lalonde and Devoe (1976), it Wolk (1973) and Lorch and Wolk (1974). must be concluded that the laminae are structural Recently, Haury and Wolk (1978) demonstrated that elements of the vesicle envelope itself and are washed mutants of ~nabaenavariabilis which were deficient in out by fixation in glutaraldehyde-osmium preparation the glycolipids of the laminated layer of the heterocyst for transmission electron microscopy which results in envelope were unable to fix dinitrogen aerobically. One the void area typically observed. of these mutants was able to fix dinitrogen if provided A striking parallel to the laminated structure of the low oxygen tensions. The authors discussed the possi- vesicle envelope in Frankia has been described in the bility that the glycolipid layers helped to provide a low For personal use only. heterocysts of several cyanobacteria (cf. Haselkorn partial pressure of oxygen within the vesicle by retrict- 1978), and we believe the laminar layer in heterocysts to ing ingress of molecular oxygen, enabling the oxygen- be structurally and functionally comparable with the labile enzyme within to function. This possibility was vesicle envelope we have described here. In their early broached earlier by Stewart (1973). ultrastructural study of the layered envelope in the The parallels between the inner laminar layer in heterocysts of Anabaena cylindrica (Fay and Lang heterocysts of Anabaena and the vesicle envelope in 1971; Lang and Fay 1971) the authors found that the Frankia include the following observations. Both layer- heterocyst envelope comprised three distinct layers: an ed structures fail to be preserved in preparations fixed outer fibrous layer, a middle homogeneous layer, and an with gl~taraldeh~de-osmiumprocessing; both are par- inner laminated layer. The inner laminated layer of the tially preserved for transmission electron microscopy if heterocyst in Anabaena is comparable with the lami- fixed by glutaraldehyde combined with oxidative post- nated layer of the vesicle envelope in Frankia. In the fixing reagents. ~othstructures show birefringence in heterocyst, the laminated layer is not preserved with polarized light, suggesting similar circumferential larni- glutaraldehyde-osmium fixation, leaving a "shrinkage nation. Using freeze-fracture preparative methods, both artefact" comparable with the void area described in the structures show multiple laminae of similar numbers and vesicles of actinorhizal nodules. Glutaraldehyde-per- thickness. In both cases, enclosure of the oxygen-labile manganate fixation did partially preserve the laminated enzyme nitrogenase can be demonstrated. Our presump- layer in the heterocyst and permitted a study of its form tion is that both serve a parallel function, probably

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by HARVARD UNIVERSITY HERBARIA on 04/04/12 and distribution. According to their view the inner involving restriction of exchange of dissolved gases. laminated layer was continuous around the entire inner We do not yet know whether the laminae in the envelope of the heterocyst with elaborated or thickened vesicles of Frankia are specialized glycolipids. In "shoulder" areas at each end of the cell at the point of preliminary experiments performed to date it has been constriction of the pore channel. In like fashion fixation possible to extract and chromatograph lipids from of vesicles cultured in vitro with glutaraldehyde follow- cultures of Frankia. This result is not unexpected, since ed by additional postfixation steps does not totally wash lipids extracted from actinomycetes have been used as a out the laminae of the vesicle but preserves them poorly. taxonomic tool within the group (e.g., Modarska and TORREY AND CALLAHAM

Modxski 1970). Further work on the lipid composition LALONDE,M., and R. KNOWLES.1975a. Ultrastmcture of the of Frankia spp. is in progress. Alnus crispa var. rnollis. Fern. root nodule endophyte. Can. J. Microbiol. 21: 1058- 1080. Acknowledgements 19756. Ultrastmcture, composition, and biogenesis of the encapsulation material surrounding the endophyte in This research was supported in part by the Maria Alnus crispa var. rnollis Fern. root nodules. Can. J. Bot. 53: Moors Cabot Foundation for Botanical Research of 1951-1971. Harvard University and research grant DEB 77-02249 of LALONDE,M., R. KNOWLES,and I. W. DEVOE. 1976. the United States National Science Foundation. Thanks Absence of "void area" in freeze-etched vesicles of the are expressed to Stanley C. Holt, Department of Alnus crispa var. rnollis Fern. root noduleendophyte. Arch. Microbiology, University of Massachusetts, for the Microbiol. 107: 263-267. use of the Balzers freeze-etch apparatus (NSF PEM LAMBEIN,F., and C. P. WOLK.1973. Stmctural studies on the 78-05656) and S. Holt and Erika Musante for technical glycolipids from the envelope of the heterocyst of Anabaena cylindrica. Biochemistry, 12: 791-798. advice in operation of the Balzers equipment. We thank . . William Newcomb, Queen's University, Ontario, for LANG,N. P., and P. FAY.1971. The heterocysts of blue-green ...... algae. II. Details of ultrastructure. Proc. R. Soc. London, ..... Fig. 1 and William Ormerod for Figs. 2-4. The authors ...... Ser. B, 178: 193-203. also express their appreciation to Alison Berry and LECHEVALIER,M. P., and H. A. LECHEVALIER.1979. The William Ormerod for cultures of CpI1. taxonomic position of the actinomycete endophytes. In Symbiotic nitrogen fixation in the management of temperate BECKING,J. H., W. E. DEBOER,and A. L. HOUWINK.1964. forests. Edited by J. C. Gordon, C. T. Wheeler, and D. A. Electron microscopy of the endophyte of Alnus glutinosa. Perry. 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Root-nodule symbioses with actinomycete- tions in developing root nodules of Cornptonia peregrina. i like organisms. In The biology of nitrogen fixation. Edited Can. J. Bot. 56: 502-531. For personal use only. i by A. Quispel. North Holland Publishing Co., Amsterdam. NICHOLS,B. W., and B. .J. B. WOOD.1968. New glycolipid : pp. 342-378. specific to nitrogen-fixing blue-green algae. Nature (Lon- ! BRYCE,T. A,, D. WELTI, A. E. WALSBY,and B. W. don), 217: 767-768. NICHOLS.1972. Monohexoside derivatives of long-chain STEWART,W. D. P. 1973. Nitrogen fixation by photosynthetic polyhydroxy alcohols: a novel class of glycolipid specific to microorganisms. Annu. Rev. Microbiol. 27: 283-3 16. heterocystous algae. Phytochemistry, 11: 295-302. TJEPKEMA,J. D. 1979. Oxygen relations in leguminous and CALLAHAM,D., W. NEWCOMB,J. G. TORREY,and R. L...... actinorhizal nodules. In Symbiotic nitrogen fixation in the ...... : : . . : : : . : . : . : : ...... :.: : . , PETERSON.1979. 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